Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts

Niada, Stefania; Giannasi, Chiara; Magagnotti, Cinzia; Andolfo, Annapaola; Brini, A.T.

doi:10.1016/j.jprot.2020.104069

“…Therefore, even though CM samples represent a complex cocktail accounting for a lot more than EVs, the presence of common freely secreted molecules between ASCs and DFs may flatten the statistical comparison and mask the impact of the observed differences on EV composition. Of note, through a differential proteomic approach, we recently gave evidence of a slightly lower similarity between the CM samples from the two cell populations in comparison to EV ones (93.4% vs. 97.2% of shared proteins between CM and EVs, respectively) (Niada et al, 2020). This discrepancy is most probably due to the contribution of non-protein molecules to the Raman profile of the samples.…”

Section: Discussionmentioning

confidence: 92%

“…Prior to Western Blot analysis, an aliquot of each CM sample was quantified through the Bio-Rad Protein Assay (Bio-Rad, Milan, Italy), while EV pellets were directly lysed in the appropriate buffer without protein quantification (Niada et al, 2020). The amount of 10 µg of CM, corresponding to ∼25 µl, and EVs derived from 1.5 × 10 6 ASCs or DFs were lysed in 5% 2-Mercaptoethanol and 2X Laemmli Buffer (Bio-Rad, Milan, Italy), separated in a 4-15% polyacrylamide gel (Bio-Rad, Milan, Italy) and transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, United States) (Niada et al, 2020).…”

Section: Western Blotmentioning

confidence: 99%

“…Prior to Western Blot analysis, an aliquot of each CM sample was quantified through the Bio-Rad Protein Assay (Bio-Rad, Milan, Italy), while EV pellets were directly lysed in the appropriate buffer without protein quantification (Niada et al, 2020). The amount of 10 µg of CM, corresponding to ∼25 µl, and EVs derived from 1.5 × 10 6 ASCs or DFs were lysed in 5% 2-Mercaptoethanol and 2X Laemmli Buffer (Bio-Rad, Milan, Italy), separated in a 4-15% polyacrylamide gel (Bio-Rad, Milan, Italy) and transferred to a nitrocellulose membrane (GE Healthcare, Chicago, IL, United States) (Niada et al, 2020). After being blocked with 5% non-fat dried milk (AppliChem, Darmstadt, Germany) and 0.1% Tween (Promega, Madison, WI, United States) in PBS, samples were probed overnight at 4 • C for the expression of Alix (NBP1-90201, 1:1,000 diluted, Novus Biologicals, Centennial, CO, United States), FLOT-1 (1:500 diluted, BD Transduction Laboratories, San Jose, CA, United States), TSG101 (T5701, 1:1,000 diluted, Millipore, Burlington, MA, United States), and CD9 (1:1,000 diluted, System Biosciences, Palo Alto, CA, United States).…”

Section: Western Blotmentioning

confidence: 99%

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Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics

Carlomagno

¹

,

Giannasi

²

,

Niada

³

et al. 2021

Front. Bioeng. Biotechnol.

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Extracellular Vesicles (EVs) and Conditioned Medium (CM) are promising cell-free approaches to repair damaged and diseased tissues for regenerative rehabilitation purposes. They both entail several advantages, mostly in terms of safety and handling, compared to the cell-based treatment. Despite the growing interest in both EVs and CM preparations, in the light of a clinical translation, a number of aspects still need to be addressed mainly because of limits in the reproducibility and reliability of the proposed protocols. Raman spectroscopy (RS) is a non-destructive vibrational investigation method that provides detailed information about the biochemical composition of a sample, with reported ability in bulk characterization of clusters of EVs from different cell types. In the present brief report, we acquired and compared the Raman spectra of the two most promising cell-free therapeutics, i.e., EVs and CM, derived from two cytotypes with a history in the field of regenerative medicine, adipose-derived mesenchymal stem/stromal cells (ASCs) and dermal fibroblasts (DFs). Our results show how RS can verify the reproducibility not only of EV isolation, but also of the whole CM, thus accounting for both the soluble and the vesicular components of cell secretion. RS can provide hints for the identification of the soluble factors that synergistically cooperate with EVs in the regenerative effect of CM. Still, we believe that the application of RS in the pipeline of cell-free products preparation for therapeutic purposes could help in accelerating translation to clinics and regulatory approval.

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“…The supernatants were then concentrated about 60 times by centrifuging at 4000 g for 90 min at 4°C in Amicon Ultra-15 Centrifugal Filter Devices with 3 kDa molecular weight cut-off (Merck Millipore, Burlington, MA, USA). This procedure allows the retention of the vesicular component of cell secretome, as previously demonstrated in [ 12 , 13 , 15 ]. The safety and efficacy of the final product obtained through this procedure have been already tested both in vitro [ 14 , 15 ] and in vivo [ 16 , 17 ].…”

Section: Methodsmentioning

confidence: 91%

Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail

Giannasi

¹

,

Niada

²

,

Morte

³

et al. 2021

Stem Cells International

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The therapeutic potential of the conditioned medium (CM) derived from MSCs (mesenchymal stem/stromal cells) in disparate medical fields, from immunology to orthopedics, has been widely suggested by in vitro and in vivo evidences. Prior to MSC-CM use in clinical applications, appropriate quality controls are needed in order to assess its reproducibility. Here, we evaluated different CM characteristics, including general features and precise protein and lipid concentrations, in 3 representative samples from adipose-derived MSCs (ASCs). In details, we first investigated the size and distribution of the contained extracellular vesicles (EVs), lipid bilayer-delimited particles whose pivotal role in intercellular communication has been extensively demonstrated. Then, we acquired Raman signatures, providing an overlook of ASC-CM composition in terms of proteins, lipids, and nucleic acids. At last, we analyzed a panel of 200 molecules including chemokines, cytokines, receptors, and inflammatory and growth factors and searched for 32 lipids involved in cell signalling and inflammation. All ASC-CM contained a homogeneous and relevant number of EVs ( 1.0 × 10 9 ± 1.1 × 10 8 particles per million donor ASCs) with a mean size of 190 ± 5.2 nm , suggesting the appropriateness of the method for EV retaining and concentration. Furthermore, also Raman spectra confirmed a high homogeneity among samples, allowing the visualization of specific peaks for nucleic acids, proteins, and lipids. An in depth investigation that focused on 200 proteins involved in relevant biological pathways revealed the presence in all specimens of 104 factors. Of these, 26 analytes presented a high degree of uniformity, suggesting that the samples were particularly homogenous for a quarter of the quantified molecules. At last, lipidomic analysis allowed the quantification of 7 lipids and indicated prostaglandin-E2 and N-stearoylethanolamide as the most homogenous factors. In this study, we assessed that ASC-CM samples obtained with a standardized protocol present stable features spanning from Raman fingerprint to specific marker concentrations. In conclusion, we identified key ingredients that may be involved in ASC-CM therapeutic action and whose consistent levels may represent a promising quality control in the pipeline of its preparation for clinical applications.

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“…Most studies on ADSC derivatives in skin rejuvenation focus on ADSC-CM. Although in proteomic analysis, ADSC-CM and ADSC-EVs show slightly different protein profiles, both ADSC-CM and ADSC-EVs contained factors related to ECM organization and immunoregulation, which are crucial for skin health [105]. Whether some agents are superior in healing efficacy is not yet settled, with contrasting results shown for different medical conditions and models.…”

Section: Theranostic/cosmetic Applications Of Extracellular Vesicles For Skin Agingmentioning

confidence: 99%

Extracellular Vesicles as Potential Theranostic Platforms for Skin Diseases and Aging

Kim

¹

,

Lee

²

,

Han

³

et al. 2021

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Extracellular vesicles (EVs), naturally secreted by cells, act as mediators for communication between cells. They are transported to the recipient cells along with cargoes such as nucleic acids, proteins, and lipids that reflect the changes occurring within the parent cells. Thus, EVs have been recognized as potential theranostic agents for diagnosis, treatment, and prognosis. In particular, the evidence accumulated to date suggests an important role of EVs in the initiation and progression of skin aging and various skin diseases, including psoriasis, systemic lupus erythematosus, vitiligo, and chronic wounds. This review highlights recent research that investigates the role of EVs and their potential as biomarkers and therapeutic agents for skin diseases and aging.

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Proteomic analysis of extracellular vesicles and conditioned medium from human adipose-derived stem/stromal cells and dermal fibroblasts

Cited by 19 publications

References 66 publications

Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics

Raman Fingerprint of Extracellular Vesicles and Conditioned Media for the Reproducibility Assessment of Cell-Free Therapeutics

Towards Secretome Standardization: Identifying Key Ingredients of MSC-Derived Therapeutic Cocktail

Extracellular Vesicles as Potential Theranostic Platforms for Skin Diseases and Aging

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